Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Zhang, Kai

Abstract

Dynein is a microtubule-based motor protein complex that generates force and drives movement toward the minus ends of microtubules. Despite extensive structural, biochemical, and biophysical studies, key questions remain about how dynein achieves its unidirectional stepping and force-generating powerstroke through its ATP-dependent mechanochemical cycle. In this dissertation, I leverage recent advances in cryo-electron microscopy (cryo-EM) and develop novel cryo-EM image processing methods to elucidate the atomic-level details of the mechanochemical cycle of full-length cytoplasmic dynein-1. I directly visualize the conformational landscape of dynein both on and off microtubules, uncovering several novel and important reactive intermediates. These findings include the discovery of a previously unknown "molecular backdoor" mechanism for phosphate release, supported by mutagenesis and single-molecule motility assays. Additionally, I conduct detailed structural analyses of dynein and propose a model to explain how dynein biases its movement toward the minus end of microtubules during its force-generating powerstroke. Extending this cryo-EM workflow to more complex axonemal dynein systems, I unexpectedly identify a novel dynein heavy chain (DHC) subfamily, DNAHX, and a new type of dynein, termed dynein-X, in sea urchin sperm. Through bioinformatics, AlphaFold structural predictions, and comparative structural analyses, I reveal the non-motile nature of DNAHX and dynein-X, providing insights into dynein evolution and function. Together, the work presented in this dissertation provides both technical advancements and biological insights, deepening our understanding of dynein’s function, mechanism, and evolution.

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